1. Field of the Invention
The present invention is directed to a microchromatographic device adapted for determination of a desired substance through rapid elution from a microcolumn, and particularly adapted for rapid determination of a relative concentration of glycosylated hemoglobin species in a red blood cell lysate of a person. The present invention is also directed to a method for rapid and reliable determination of the relative concentration of glycosylated hemoglobins in the red blood cell lysate of a person.
2. Brief Description of the Prior Art
It has been known in the prior art that several, chromatographically separable minor hemoglobin species are present in the red blood cell lysate of human beings. Glycosylated minor hemoglobin species customarily designated HB-A.sub.1a, Hb-A.sub.1b and Hb-A.sub.1c are believed to be Amadori rearranged Shiff base type condensation products of glucose and hemoglobin, wherein a hexose derived from glucose is attached to an N-terminal amino acid in the .beta. peptide chain of hemoglobin. The reaction between glucose and the N-terminal amino acid is understood to be practically irreversible under physiological conditions. Therefore within the average 120 day life span of human red blood cells the relative concentration of glycosylated hemoglobin species when compared to non-glycosylated hemoglobin species provides a good numerical indication of the time averaged blood sugar level of a person. Stated differently, the relative concentration of glycosylated hemoglobin species in the red blood cell lysate of a person acts as a numerical index of the cumulative blood sugar history of the person for the preceding 3-4 month time period.
For a detailed description of the chemical nature of glycosylated hemoglobins and their diagnostic significance particularly in monitoring patients suffering from diabetes mellitus, reference is made to the following publications: Kenneth H. Gabbay, Karen Hasty, Jan L. Breslow, R. Curtis Ellison, H. Franklin Bunn, and Paul M. Gallop; Glycosylated Hemoglobins and Long-Term Blood Glucose Control in Diabetes Mellitus, Journal of Clinical Endocrinology and Metabolism, Volume 44 pages 859-864 (1977); H. Franklin Bunn, David N. Haney, Steven Kamin, Kenneth H. Gabbay and Paul M. Gallop: The Biosynthesis of Human Hemoglobin A.sub.1c Slow Glycosylation of Hemoglobin in Vivo, The Journal of Clinical Investigation, volume 57, pages 1652-1659 (1976).
In recognition of the practical diagnostic significance of determining the relative glycosylated hemoglobin concentration of red blood cell lysates obtained from patients, the prior art has developed a plurality of microchromatographic techniques and devices for conducting such determinations.
These techniques and devices involve a miniature or microcolumn having a packing of a suitable ion exchange resin. A sample of hemolysed red blood cells taken from a specific person is placed on a top end of the microcolumn, and a first buffer solution is passed through the microcolumn. The first buffer solution is chosen to selectively elute glycosylated hemoglobin species only, while nonglycosylated hemoglobin species stay adhered to the ion exchange resin in the microcolumn. A first numerical value proportional to the concentration of the glycosylated hemoglobin species in the collected eluate of the first buffer is determined spectrophotometrically.
In order to determine a second numerical value proportional to the sum of the concentration of glycosylated and nonglycosylated hemoglobin species in the hemolysate of the specific person, the prior art has employed two alternative approaches. According to a first approach, a second buffer solution is used to elute from the microcolumn the nonglycosylated hemoglobin species, and a third numerical value proportional to its concentration is determined spectrophotometrically. Summation of the first and third numerical values provides the second numerical value. According to a second approach, red blood cells of the patient are lysed in a sample separate from the microcolumn, and the second numerical value is determined spectrophotometrically, after appropriate dilution if necessary, in that sample. A simple ratio of the first and second numerical values, taking dilutions into account if applicable, expressed in a percentage form provides the diagnostic number or index which is characteristic of the history of the patient's blood sugar level.
U.S. Pat. Nos. 4,142,855; 4,142,856; 4,142,857 and 4,142,858 and the publication: Rapid Estimation (21/2 Hours) of Glycosylated Hemoglobin for Routine Purposes by P. A. Kynock and H. Lehmann, The Lancet July 2, 1977 pages 16-17, are exemplary of the above summarized microchromatographic techniques and devices. These patents and the publication describe the specific nature and parameters of the materials used in the techniques.
As it is readily appreciated by those skilled in the diagnostic arts, routine diagnostic determinations performed in a laboratory of a clinic or hospital should preferably be completed in a time span of minutes rather than hours. Furthermore, diagnostic measurements strive for increasing accuracy and reliability.
In this regard, it is noted that the prior art techniques of determining the diagnostic number or index characteristic of the history of the blood sugar level of a person are, generally speaking, quite temperature dependent. In other words, reproducibility of the results is impaired if several measurements are not conducted at essentially identical temperatures.
An article authored by R. E. Davis and D. J. Nicol titled "A Rapid Simplified Method for Routine Measurement of Glycosylated Hemoglobin," The Lancet Aug. 12, 1978 pages 350-351, describes a microchromatographic method wherein a buffer solution capable of eluting the glycosylated hemoglobin species from the microcolumn is rapidly forced through the microcolumn in a centrifugation step. As a result, the diagnostic measurement is said to be completed in approximately 5 minutes.
In another effort to simplify the diagnostic glycosylated hemoglobin determination procedures, a diagnostic microcolumn was recently made commercially available in the United States wherein lysis of the red blood cells is performed in an upper reservoir of the microcolumn rather than in a separate vessel.
In spite of the above described advances in the prior art, there is still significant room for improvement to render these diagnostic determinations easier to perform, faster and less temperature dependent. The present invention provides such a technique and a microchromatographic device to perform the technique.